This invention relates to novel folic acid derivatives useful for determining the amounts of folic acid and its metabolites in biological or other liquids by radioassay, and to methods of analyzing such biological liquids using the materials disclosed herein.
The usefulness of serum and red cell folate assays in the diagnosis of nutritional anemias and related illnesses is now well established. Some of the primary conditions where folate levels have diagnostic significance include nutritional deficiencies, especially in cases of severe alcoholism, and a variety of malabsorption syndromes including functional damage to the upper third of the small bowel, pregnancy, and a number of types of megaloblastic anemia. Folic or pteroylglutamic acid as such is not biologically active in man. This material undergoes conversion in the body, and the dominant form in human serum is N,5-methyltetrahydrofolic acid.
Early methods of analyzing endogenous folates involved a microbiological assay using Lactobacillus casei, whose growth is dependent on the presence of folic acid and its derivatives in an incubation medium. However, this technique is cumbersome, time consuming, and subject to interference by drugs such as folate antagonists or antibiotics. Recent developments in radioassay techniques have resulted in the development of competitive binding methods for the determination of folates which overcome many of the limitations of the microbiological procedures. The basic principle of such methods is that of competitive protein binding. Basically, advantage is taken of the affinity of folates to specific folate binders, i.e., binders which specifically bind only folates. Suitable binders are known and are obtainable from sources such as cow's milk, hog's kidney, etc. Folate labeled with radioactivity is mixed with the sample to be analyzed and a specific folate binder, and the radioactive labeled folate competes with the unlabeled folates in the sample to be analyzed for binding by the folate-specific binder. As a result, the ratio of bound-labeled folic acid to free-labeled folic acid diminishes as the concentration of unlabeled folate is increased. Accordingly, the concentration of folate in an unknown sample, e.g., a patient's blood serum, is obtained by comparing inhibition of labeled folate binding observed, with that produced by known amounts of folate, as presented in a standard curve. After mixing the labeled folate, the sample and the binder, the bound material, both labeled and unlabeled, is separated from the unbound or "free" material, by any of a variety of known separation techniques, e.g., by adsorption onto charcoal or other known adsorbents, or by precipitation techniques, e.g., using ammonium sulfate or polyethylene glycol or other known precipitants, or by dialysis. The radioactivity in the bound fraction is then measured, the data is plotted on a graph to prepare the standard dose-response curve, and the amount of folate in a sample is determined by interpolation from the standard curve.
Thus, the use of protein binding in folate determination requires the use of a binder, a "tracer," which is a radioactive-labeled material which has the ability to compete with unlabeled material for binding, and a "standard." The standard is the folate material which is added in known specified amounts to the binder and the tracer, in order to obtain the standard curve.
Since the predominant form of folate found in human serum is the reduced 5-methyl derivative of folic acid, early competitive binding techniques utilized the N,5-methyltetrahydrofolic acid as the standard, against which the biological samples would be compared. The tracer used was generally tritiated folic acid, and the binder used was generally one obtained from milk. Such procedures suffered from a number of disadvantages, caused in part by the extreme instability of the N,5-methyltetrahydrofolic acid used as a standard, and also from the disadvantages of using tritium as the labeling radionuclide. Tritium requires cumbersome liquid scintillation counting procedures and is generally unsuitable for radioassay techniques, since its use is subject to a variety of errors. See, Chervu et al., "Radiolabeling of Antigens: Procedures and Assessment of Properties," Seminars in Nuclear Medicine, 5:157, 158 (1975). The preferred labeling radionuclide is radioactive iodine, more preferably .sup.125 I. However, incorporation of the large iodine atom into the molecular structure of the material to be analyzed changes that structure, and can have adverse affects on its affinity for the binder. One approach that has been taken to overcome this difficulty is conjugation labeling, in which a molecule labeled with .sup.125 I is added to (conjugated with) the molecule which is to be determined, in order to make a composite molecule in which the iodine will be removed from the situs of attraction to the binder and will, therefore, not interfere with the affinity of the tracer to the binder.
It is an object of this invention to provide novel folic acid derivatives which are useful in a radioassay for folic acid and its metabolites in biological liquids.
It is a further object of this invention to provide a method of radioassaying for folic acid and its metabolites in biological liquids, using novel folic acid derivatives.
It is a further object of this invention to provide a method for radioassaying which is easy, inexpensive, and reproducible, yielding labeled tracers having high specific binding activity, specific radioactivity, purity, and extremely long shelf life.
The folic acid derivatives of this invention are conjugates of folic acid with histamine, or with iodinated histamine, and are represented by the following general structural formula: ##STR2## in which R is hydrogen or radioactive iodine, such as .sup.125 I or .sup.131 I. As is known in the art, it is possible to have a plurality of glutamate linkages attached to the pteroyl moiety, and in such cases the labeled or unlabeled histamine moiety would bond to the pteroyl moiety by such linkages.
In accordance with the present invention, it has been found that the labeled folic acid-histamine conjugate of the present invention is a particularly effective tracer in folic acid determinations by radioassay, and that such tracer has an extremely long shelf life, as compared with other folic acid tracers. This is particularly important, because, although use of radioactive iodine as the labeling radionuclide is highly advantageous from the standpoint of providing higher specific activity, and thus higher sensitivity in shorter counting times, low chemical stability of the tracer compound can substantially irradicate the advantages, from a practical standpoint, since if all of the tracer rapidly chemically degrades, the tracer will no longer compete with the material to be measured for the binder. The folic acid-histamine conjugate of the present invention, although preferably stored at referigerator temperature (2.degree.-8.degree. C.), is so stable that it can be simply stored at room temperature for extended periods without substantial degradation, whereas tritiated folic acid, for example, is unstable to the extent that it must normally be stored away from light and frozen in separate aliquoted tubes, to avoid any thawing and refreezing of unused portions of that tracer.
It has also been discovered that unlabeled folic acid-histamine conjugate can be used to distinct advantage as a standard in the radioassay process for determination of folic acid and its metabolites in biological or other liquids. It is far more stable than N,5-methyltetrahydrofolic acid previously used as a standard, and under suitable conditions gives essentially identical results. Optimal conditions for such use can be easily determined by known methods, e.g., that disclosed in Givas et al., "pH Dependence of the Binding of Folates to Milk Binder in Radioassay of Folates," Clinical Chemistry, 21:427-8 (1975), incorporated herein by reference.
The compounds of this invention may be simply prepared by reacting folic acid with histamine in the presence of a coupling reagent, e.g., a a dehydrating reagent such as a carbodiimide, in aqueous medium. Preferably the reaction is conducted at a pH of about 6 to 10, most preferably at a pH of about 7.5 to 9. Suitable coupling reagents include a wide variety of compounds commonly used in coupling procedures in peptide or other syntheses, including dicyclohexyl carbodiimide, 1-ethyl-3-(3-morpholinomethyl) - carbodiimide, N,N'-carbonyldiimidazole, N,N'-carbonyl-5-triazine, bis-5-phenylene pyrophosphite, diethyl chlorophosphonate, diethyl cyanamide, diethyl ethylenepyrophosphite, diphenylketene p-tolylamine, ethoxyacetylene, ethylene chlorophosphite, N-ethyl-5-phenyl-isoxazolium-3'-sulfonate N-hydroxypyridine, N-hydroxyphthalimide, N-hydroxypiperidine, N-hydroxysuccinimide, phenylphosphorodi-(1-imidazolate), and still others will be readily recognized by those skilled in the art. Separation of the folic acid-histamine conjugate from the reaction mixture is by standard chromatographic or other separation techniques.
The molar ratios of histamine to folic acid to coupling reagent may vary over wide ranges, and may generally be from about 1:1:1 to about 3:1:20 or 1:3:20. Preferably the coupling reagent is added in molar excess, being at least 0.1 moles in excess of the lesser of the other two reactants. The reaction can be run at 0.degree. C. to 30.degree. C. and should be permitted to proceed from about 1 to 72 hours, preferably being run for approximately 1 day at room temperature.
Other methods of producing the conjugate willbe readily apparent to those skilled in the art, including the mixed anhidride technique, e.g. that disclosed in U.S. Pat. No. 3,989,812, issued Nov. 2, 1976, to Barrett et al.
Iodination is most conveniently carried out by the Chloramine T reaction, although other oxidation techniques are also well known. See, for example, Chervu, supra, incorporated herein by reference.
The method of use of the compounds of the present invention, both as tracer and as standard, in conventional radioassay techniques, will be readily apparent to the skilled in the art. The standard, which may be folic acid-histamine conjugate of the present invention, or may be N,5-methyltetrahydrofolic acid or folic acid per se, is generally prepared by the type of procedure known in the art wherein standard samples are prepared having various concentrations of the standard. The standard and patient samples are each mixed with a buffer, and the resulting buffered solutions are then normally heated or otherwise appropriately treated to release folates from any binders in the serums, red blood cells or other biological liquids being tested. After cooling to room temperature, the tracer solution and the binder are mixed with the standard and samples, and the mixtures are incubated for a sufficient amount of time to permit completion of binding of the tracer and other folates in the various tubes. This incubation is preferably conducted at a pH range which depends primarily on the type of standard being used. When N,5-methyltetrahydrofolic acid is the standard, the pH range is not critical, and preferably approximately matches the pH of the biological fluid being employed, preferably a pH of about 6.5 to 8.5. When folic acid is the standard, however, the pH is highly critical, and must be within the narrow range of about 9.2 to 9.4. The incubation is conducted at a temperature of 0.degree. to 50.degree. C., most preferably about 15.degree. to 25.degree. C. After the completion of incubation, the bound folates are separated from the free folates, e.g., by adsorption with a suitable adsorbent, e.g., a charcoal adsorbent. The supernatant (bound) folates are determined by counting in a gamma counter, generally for a period of one minute per tube at the usual counting efficiencies of 50 to 80%. A standard curve is prepared, e.g., by plotting the average net counts for each standard against the corresponding concentration of folic acid in ng/ml for each standard tube, and the concentration of folate in the sample tubes containing the biological liquid being analyzed is determined by interpolation from the standard curve.
In a particularly advantageous embodiment of the present invention, there is provided a kit for the entire assay of folate samples, which kit includes a folate assay buffer, for maintaining the pH of the desired level for comparability of binding between the standard and the folates in the samples to be measured, a folate binder, preferably .beta.-lactoglobulin, the folic acid-histamine conjugate [.sup.125 I] tracer, and the folic acid standards. Preferably the kit also contains other components for the analysis, e.g., a charcoal suspension usable to separate the bound from the free folic acid, and a folate control serum, which comprises a human serum with a nominal folic acid concentration, and which can be used as a separate check on the assay system. Preferably the kit also contains ascorbic acid tablets for preservation of the sera samples prior to and during assay.